There is increasing interest in the production of important biologicals by animal cell culture using more efficient technology to meet demands in quantity, purity and safety for these products, preferably at reduced unit cost. Scale-up of the traditional batch culture has in practice several technical and economic limitations resulting from the low product titer and poor volume productivity. An alternative method is intensification of the reaction process by recycling or retaining cells in the bioreactor. In such cultures, cells can be maintained at high density through a retention system that can allow cells to be perfused with fresh medium while withdrawing spent medium at the same time. In this context of high density culture, oxygen is not only essential for cell growth but also for the maintenance of cell viability and need to be delivered gradually in function of cell demand. Even within the range of oxygen concentrations sufficient for cell survival, the concentration of oxygen has a profound influence on cell signaling, growth factor production, growth and differentiation (Muschler et al. 2004). Excess levels of dissolved oxygen may enhance the formation of superoxide radicals, peroxides and hydroxyl radicals, which can damage the DNA and cell membranes, reduce cell viability, induce cell death and create the condition of “oxidant stress” (Ellis 1991; Cacciuttolo et al. 1992). The inhibition of cell growth in such conditions may be due to the formation of peroxides and free radicals, which “down-regulate” cell proliferation, reduce cell viability and create the condition of “oxidant stress” (Ellis 1991). These oxidising agents are responsible for damaging cellular materials such as DNA, carbohydrates and proteins. An elevated dissolved oxygen level has also been shown to prolong cell-cycle duration by inhibiting the initiation of DNA synthesis (Barlin et al. 1979). More recently, Cacciuttolo et al. (1992) provided evidence that dissolved oxygen concentrations higher than 100% caused increased DNA strand breaks as well as affecting metabolic functions such as glucose consumption rate, lactate production rate and cell growth. Typical demands for oxygen by animal cells during batch culture are in the range 2×10−16-2×10−15 mol O2 cell−1 min−1. It is not difficult to cope with this oxygen requirement in low-cell-density batch or continuous cultures, at least for small scales of operation, but meeting the oxygen demand in high-cell-density perfusion cultures is a serious problem. In addition, enhancing bacteria and cell growth represents an economic challenge for bioindustry.
Different approaches have been developed to supply oxygen in cell cultures. In general, the growth and/or culture of animal cells, especially mammalian cells requires a constant supply of oxygen and effective removal of gaseous metabolic by-products, mainly carbon dioxide. The requisite gas exchange can be accomplished in a number of ways, including agitation of the reaction vessel and bubbling of oxygen-containing gases through the culture. In order for agitation to be effective, very low volumes of culture medium must be used since the effectiveness of this method depends on exposure of all of the cells in the culture to the surface. Bubbling of gases obviates this problem to some extent, but superimposes another wherein sufficient bubbling to be effective creates shear forces believed to be harmful to the relatively delicate animal cell membranes.
Another approach to supply oxygen in a fermentation vessel for animal cells consists in using a tube or hose of a synthetic polymer, such as silicone rubber, laminated silicone rubber products, or a polytetrafluoroethylene (Teflon®), to provide oxygen through diffusion (U.S. Pat. No. 4,649,114). This tube is required to be non adherent with regard to the cells, thick enough to provide mechanical strength, and thin enough to permit oxygen to pass through readily.
Another examples of devices allowing the supply of oxygen through a membrane are the hollow fiber systems (FiberCell®, Cellco®, Technomouse® from Integra Biosciences), which rely on the supply of oxygen along with culture medium through a closely spaced series of hollow fibers interspersed among the suspended cell culture. One drawback of this hollow fiber system is that the cells have to be in close proximity to the fibers to be supplied in oxygen.
The present invention aims to provide a new generation of bioreactor capable of delivering oxygen to cell culture using oxygen carrying molecules. In this new generation of bioreactor, oxygen can be delivered to the cells progressively according to the biomass growth avoiding stress and cell damages linked to oxygen excess.
Particularly, the present invention aims to provide a new generation of bioreactor capable of delivering oxygen to cell culture by means of oxygen carrying molecules, without using any cofactor.